Analysis of Antimicrobial Resistance Genes (ARGs) in Enterobacterales and A. baumannii Clinical Strains Colonizing a Single Italian Patient

The dramatic increase in infections caused by critically multidrug-resistant bacteria is a global health concern. In this study, we characterized the antimicrobial resistance genes (ARGs) of K. pneumoniae, P. mirabilis, E. cloacae and A. baumannii isolated from both surgical wound and rectal swab of a single Italian patient. Bacterial identification was performed by MALDI-TOF MS and the antimicrobial susceptibility was carried out by Vitek 2 system. The characterization of ARGs was performed using next-generation sequencing (NGS) methodology (MiSeq Illumina apparatus). K. pneumoniae, P. mirabilis and E. cloacae were resistant to most β-lactams and β-lactam/β-lactamases inhibitor combinations. A. baumannii strain was susceptible only to colistin. The presence of plasmids (IncN, IncR, IncFIB, ColRNAI and Col (MGD2)) was detected in all Enterobacterales but not in A. baumannii strain. The IncN plasmid and blaNDM-1 gene were found in K. pneumoniae, P. mirabilis and E. cloacae, suggesting a possible transfer of this gene among the three clinical species. Conjugation experiments were performed using K. pneumoniae (1 isolate), P. mirabilis (2 isolates) and E. cloacae (2 isolates) as donors and E. coli J53 as a recipient. The blaNDM-1 gene was identified by PCR analysis in all transconjugants obtained. The presence of four different bacterial species harboring resistance genes to different classes of antibiotics in a single patient substantially reduced the therapeutic options.


Introduction
Antimicrobial resistance represents one of the most serious global public health issues [1]. Several microorganisms known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have emerged as globally critical multidrug-resistant (MDR) pathogens requiring continues monitoring and development of new drugs [2,3]. To date, only few drugs in development are potentially active against ESKAPE pathogens [4]. Among these, the Gram-negative P. aeruginosa, K. pneumoniae and other Enterobacterales are of great concern for their high level of resistance to most antibiotics and, in particular, to carbapenems, often considered as our last line of defense against critical pathogens [5][6][7]. In the last decade, infections caused by carbapenemase-producing Enterobacterales (CPE) have dramatically increased [6][7][8][9][10][11]. In particular, the KPC and NDM variants are the most widespread carbapenemases in clinical strains with a wide range of infections [12][13][14][15].
This study reports a detailed characterization of the antimicrobial resistance genes (ARGs) in P. mirabilis, K. pneumoniae, E. cloacae and A. baumannii, isolated from surgical wound (SW) and rectal swab (RS) of a particular hospitalized patient.

Antimicrobial Susceptibility
Overall, six clinical isolates obtained from SW and RS were analyzed against a large panel of antibiotics. As given in Table 1, the E. cloacae and K. pneumoniae isolates showed resistance to carbapenems (ertapenem, imipenem and meropenem), cephalosporins (cefotaxime, ceftazidime and cefepime), amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftolozane-tazobactam and ceftazidime-avibactam. However, E. cloacae and K. pneumoniae isolates were susceptible to amikacin, gentamycin and colistin. A similar susceptibility profile was found for P. mirabilis isolates. The A. baumannii strain was susceptible only to colistin.  The draft genome of the six isolates recovered for this study was obtained using MiSeq Illumina platform. The sequence analysis revealed that the total number of sequenced nucleotides was 4.6-4.8 Mb for E. cloacae isolates, 3.7-4.3 Mb for P. mirabilis isolates, 5.7 Mb for K. pneumoniae and 3.7 Mb for A. baumannii ( Table 2). The MLST of K. pneumoniae, E. cloacae and A. baumannii indicates that these strains belonged to the lineages ST4587, ST45 and ST2, respectively. To date, molecular characteristics and genetic relationships among Proteus spp. have not been elucidated, and for this reason, the MLST analysis of P. mirabilis was not launched.

Conjugation Experiments and PCR Assays
Conjugation experiments were performed using E. coli J53 strain as a recipient and P. mirabilis (RS and SW), E. cloacae (RS and SW) and K. pneumoniae RS as donors. Conjugational transfer of meropenem resistance was ascertained in all three systems, and the presence of bla NDM-1 was confirmed by PCR in all transconjugants obtained.

Discussion
Here, we described the molecular characterization of ARGs of K. pneumoniae, E. cloacae, P. mirabilis and A. baumannii clinical strains isolated from different sample sites of a single hospitalized patient. The case was not epidemiologically related to other hospitalized patients, and no information was available about the stay of the patient in LTCF. The characterization of ARGs was performed by NGS analyzing the resistome of all strains. On the basis of the draft genome analysis, we have noted that the IncN plasmid was found in all Enterobacterales analyzed. The IncN belongs to a broad-host-range plasmids with a size of 30 to 70 Kb that, often, carry a great variety of resistance genes, including bla CTX-M , bla VIM and bla NDM [16][17][18][19]. In this study, IncN was simultaneously present with IncQ1 in P. mirabilis, with IncFIB and ColRNAI in E. cloacae and with IncR in K. pneumoniae. The IncQ1 belongs to the MOBQ group with a medium-small size (8)(9)(10)(11)(12)(13)(14) and, often, they carry sul-strA-strB gene cluster [18]. The IncR (40-160 Kb) is a mobilizable plasmid frequently cointegrated with IncN plasmid [19], the same cluster found in P. mirabilis and E. cloacae (this study). The β-lactams resistance genes found in K. pneumoniae, P. mirabilis and E. cloacae were bla NDM-1 , bla ACT-15 , bla LEN-22 and bla TEM-1B . However, only bla NDM-1 was found in all Enterobacterales, and for this reason, we have supposed that IncN plasmid harbored bla NDM-1 gene. On the basis of conjugation experiments, we have presumed the circulation of bla NDM-1 gene via IncN plasmid among K. pneumoniae, P. mirabilis and E. cloacae isolated from the single patient. The wide distribution of bla NDM-1 and its natural variants among clinical and community-acquired Enterobacterales is related to the fact that they can be carried by different plasmid types (IncA/C, IncF, IncL/M or untypable) that are readily self-transmissible by conjugation [20]. The bla NDM promiscuity is related to its high mobilization capacity into plasmids or chromosomes [21]. Patients simultaneously infected and/or colonized with multiple species of CPE are more frequently observed [22][23][24]. Several cases of interspecies exchange of identical bla KPC-, bla OXA-48− and bla NDM-1 -carrying plasmids have been described [22][23][24][25][26]. In particular, those involving the bla NDM-1 were mainly related to the horizontal spread of broad-host-range IncC plasmids (formerly, IncA/C2) [26]. Invasive infections by MBL-producing Enterobacterales are associated with high mortality rates (>30%), especially, in the hospital setting when critically ill patients are involved [27,28]. The spread of CPE is significantly increasing in healthcare settings and, also, in long-term care facilities (LTCFs) [15][16][17]. The draft genome analysis of A. baumannii strain, analyzed in this study, exhibited the presence of bla OXA-23 , bla OXA-66 and bla ADC-25 genes. It is very common to find the simultaneous presence of bla OXA-23 and bla OXA-66 in the genome of carbapenem-resistant A. baumannii strains [29,30]. The OXA-66 is an OXA-51-like enzyme, intrinsically overexpressed in A. baumannii strains, which is able to confer high resistance to carbapenems [31]. The bla ADC genes are also chromosomally encoded in A. baumannii strains [32]. The presence of ISAba24 and ISAba26 upstream the bla OXA genes indicates the plausible presence of a strong promoter that drives expression of the downstream genes and facilitates the transferability of resistance determinants [33,34]. In particular, the LTCFs represent an important ARGs' reservoir in older resident people who are more vulnerable to bacterial infections due to multiple chronic illnesses.

Clinical Case Description
In September 2020, an 88-year-old woman was admitted to the Emergency Department of the A. Manzoni Hospital (Lecco, Italy) following an accidental fall. The X-ray revealed a displaced fracture of the left femur involving the lesser trochanter. The patient's medical history revealed previous fractures of the same femur, presumed autoimmune liver disease, diabetes, obesity and lower limb polyneuropathy. No recent hospitalization was reported. Four days after admission, the patient underwent surgery after washing the fracture site. One week after surgery, the patient was discharged from the Orthopedics unit to an LTCF; but a few days later, she was newly admitted to the General Medicine of the A. Manzoni Hospital, showing hypotension and diffuse icterus. Based on the hospital protocol for patients coming from LTCF, a rectal swab for CPE screening was performed, whereas an antimicrobial treatment was empirically initiated with amoxicillin/clavulanic acid (0.625 g, tid). Laboratory data showed increased values of lipases, bilirubin, AST and ALT enzymes, thus suggesting hepatic dysfunction. Based on appropriate imaging, acute cholecystitis with gallbladder hydrops was diagnosed and empiric therapy was then changed to piperacillin/tazobactam (4.5 g, tid) and gentamicin (240 mg, once a daily). Bacterial isolates were recovered from MacConckey agar (bioMérieux, Marcy l'Etoile, France), after an 18-22 h incubation period in aerobic conditions (37 • C) in the context of a laboratory clinical routine. In particular for rectal swabs, bacterial isolates resistant to carbapenems were also recovered from chromogenic Brilliance CRE agar (Thermo Fisher Scientific, Waltham, MA, USA). Cultures from SW were positive for E. cloacae complex SW and P. mirabilis SW, both producing an NDM-type carbapenemase. The RS performed at the same time showed positive for E. cloacae complex RS, P. mirabilis RS and K. pneumoniae isolates, all of them producing an NDM-type enzyme. Subsequently, a carbapenem-resistant A. baumannii SW was also isolated from the wound. The patient was discharged after 45 days in good health conditions, and a home care regimen was assessed.

Strains Identification and Antibiotic Susceptibility Testing
The bacterial identification was performed by MALDI-TOF mass spectrometry (Vitek MS, bioMérieux, Marcy l'Étoile, France). The antimicrobial susceptibility was determined using both the Vitek 2 system (bioMérieux, Marcy l'Étoile, France) and the Sensititre™ Gram Negative Panel (ThermoFisher, Waltham, MA, USA). Susceptibility results were interpreted according to current EUCAST criteria. Carbapenemase production was first assessed using phenotypic methods, including an immunochromatographic assay (RESIST-4 O.K.N.V., Coris Bio-Concept, Gembloux, Belgium) and specific inhibitor disks (KPC+MBL Confirm ID Kit, Rosco Diagnostica), Figure S1. The clinical strains analyzed in this study were from the surgical wound (E. cloacae SW, P. mirabilis SW, A. baumannii SW) and rectal swab (E. cloacae RS, P. mirabilis RS, K. pneumoniae RS) samples.

Conjugation Assays
Conjugation experiments were performed using Escherichia coli J53 (rifampicin-resistant strain) as a recipient and P. mirabilis (RS and SW), E. cloacae (RS and SW) and K. pneumoniae RS strains as donors. Transconjugants were selected on Luria-Bertani (LB) agar plates supplemented with 300 mg/L rifampicin and 2 mg/L meropenem or 100 mg/L ampicillin. The detection sensitivity of the assay was ≥5 × 10-7 transconjugants per recipient.

PCR Experiments
One colony of each transconjugant was picked, dissolved in 100 µL of sterile H 2 O and boiled at 100 • C. The mixture was harvested at 14,000× g, and 2 µL of supernatant was used for PCR experiments with specific NDM-1 primers. Primers and PCR conditions were from our previous study [40].

Conclusions
In this study, we have characterized the ARGs from four different bacterial species isolated from a single patient, who was admitted to the Orthopedics unit of the A. Manzoni Hospital. The patient was then discharged from the Orthopedics to an LTCF. After a few days, the patient was newly admitted to the General Medicine of the same hospital for different problems. This represents a clear example of bacterial pathogens spreading from community to hospital settings and vice versa. Unfortunately, we have no information about the epidemiological situation of the LTCF that accommodated the patient. Results obtained in this study showed the persistence of bla NDM-1 in three different Enterobacterales species isolated from a single patient. NDM producers are not commonly related to the Italian epidemiologic context, but they are emerging and increasingly reported [41,42]. Moreover, infections caused by pathogens harboring ARGs that confer resistance to different classes of antibiotics substantially reduced the therapeutic options, especially, when bacteria harbored MBLs.